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Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...

The Ohio State University College of Pharmacy

The Ohio State University Comprehensive Cancer Center

Peroxynitrite (PNT) is a highly reactive oxidant that plays a key role in the destruction of foreign pathogens by specific phagocytic immune cells such as macrophages. However, when its production is dysregulated, this oxidant can contribute to cardiovascular disease, neurological diseases, and cancer. To facilitate the detection of PNT in living cells, we designed and synthesized a fluorescent sensor termed PS3 that accumulates in membranes of the endoplasmic reticulum (ER). This subcellular targeting enhances the proximity of PS3 to the phagosome of macrophages where PNT is generated. When PS3-treated macrophages are stimulated with 10 µm opsonized tentagel microspheres, antibody-dependent cellular phagocytosis (ADCP) of these particles results in production of endogenous PNT, oxidative cleavage of the fluorescence-quenching phenolic side chain of PS3, and increased fluorescence that can be detected by confocal laser scanning microscopy, flow cytometry, and other assays. We describe methods for the synthesis of PS3 and evaluation of its photophysical properties, selectivity, and reactivity. We further report differential production of PNT during ADCP by the phagocytic cell lines RAW 264.7, J774A.1, and THP-1, as detected by confocal microscopy and changes in fluorescence intensity on 96-well plates. This approach may be useful for identification of modulators of PNT and related studies of ADCP.

Fluorescent detection of peroxynitrite during antibody-dependent cellular phagocytosis.

Taxoids such as paclitaxel (Taxol) are an important class of anticancer drugs that bind β-tubulin and stabilize cellular microtubules. To provide new chemical tools for studies of microtubules, we synthesized derivatives of paclitaxel modified at the 7-position with the small coumarin-derived fluorophore Pacific Blue (PB). Three of these Pacific Blue-Taxoids termed PB-Gly-Taxol, PB-β-Ala-Taxol, and PB-GABA-Taxol bind purified crosslinked microtubules with affinities of 34-265 nM, where the affinity can be tuned based on the length of an amino acid linker. When added to living cells in the presence of verapamil or probenecid as inhibitors of efflux, these compounds allow visualization of the microtubule network by confocal microscopy. We describe methods for the synthesis of these probes, determination of their affinities for crosslinked tubulin, and imaging of microtubules in living HeLa cells. We further describe their uptake by Caco-2 cells and two transporter-deficient Caco-2 knockout cell lines in the absence and presence of efflux inhibitors by flow cytometry. These studies revealed that p-glycoprotein (MDR1) and multidrug-resistance protein 2 (MRP2) are major mediators of efflux of these molecular probes. These compounds provide useful tools for studies of microtubules and cellular efflux transporters in living cells.

Pacific Blue-Taxoids as Fluorescent Molecular Probes of Microtubules.

Fluorinated analogues of the fluorophore pyronin B were synthesized as a new class of amine-reactive drug-like small molecules. In water, 2,7-difluoropyronin B was found to reversibly react with primary amines to form covalent adducts. When this fluorinated analogue is added to proteins, these adducts undergo additional oxidation to yield fluorescent 9-aminopyronins. Irradiation with visible blue light enhances this oxidation step, providing a photochemical method to modify the biological properties of reactive amines. In living HeLa cells, 2,7-difluoropyronin B becomes localized in mitochondria, where it is partially transformed into fluorescent aminopyronins, as detected by spectral profiling confocal microscopy. Further excitation of these cells with the blue laser of a confocal microscope can depolarize mitochondria within seconds. This biological activity was only observed with 2,7-difluoropyronin B and was not detected with analogues such as pyronin B or 9-methyl-2,7-difluoropyronin B. This irradiation with blue light enhances the cellular production of reactive oxygen species (ROS), suggesting that increased ROS in mitochondria promotes the formation of aminopyronins that inactivate biomolecules critical for maintenance of mitochondrial membrane potential. The unique reactivity of 2,7-difluoropyronin B offers a novel tool for photochemical control of mitochondrial biology.

Blake R. Peterson

Division of Medicinal Chemistry and Pharmacognosy

The Ohio State University College of Pharmacy

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography